This invention relates to the control of prokaryotic multidrug susceptibility. More specifically, this invention relates to the use of oligonucleotides for the treatment of diseases, disorders, and conditions associated with drug resistance in bacteria.
Bacterial antibiotic and antimicrobial resistance has been recognized since the advent of antimicrobial agents. In the past, the appearance of resistant microorganisms has been addressed by the continued availability of effective alternative drugs. As reported by Neu (Science (1992) 257:1064-1073), the situation has recently changed dramatically, leading to increasing morbidity and mortality. The growing number of pathogens resistant to multiple, structurally unrelated drugs, and the fact that no new class of antimicrobials is likely to be introduced before the end of the decade, have been blamed for the present crisis of clinical and epidemiologic significance (see, e.g. Neu, supra). Thus, as discussed extensively in the medical and scientific literature, there is a growing need to formulate effective therapeutic approaches to counter the emergence of novel bacterial strains resistant to antibiotics.
Resistance to an antimicrobial agent may be an inherent property of the infecting organism, or may result from mutation or from transfer of an extrachromosomal genetic determinant, such as plasmids and transposons, followed by selection of resistant organisms. In recent years there has been increased interest in the role of chromosomal sequences involved in conferring antibiotic resistance. A novel chromosomal stress response locus, the multiple antibiotic resistance (mar) locus has been shown to control the expression of chromosomal genes involved in intrinsic multidrug susceptibility/resistance to multiple, structurally different antibiotics and other noxious agents in Escherichia coli and in other members of the Enterobacteriaceae family (Cohen et al. (1988) J. Bacteriol. 170:5416-5422; Cohen et al. (1993) J. Infec. Dis. 168:484-488).
The mar locus has been reported to include two transcriptional units, marC and marRAB. Each unit is divergently transcribed from a central regulatory region, marO (Cohen et al. (1993)J. Bacteriol. 175:1484-1492; and Goldman, J. et al. (1996) Antimicrobiol. Agents Chemo. 40:1266-1269). Both operons, marORAB and marC are necessary for the full expression of the Mar phenotype. Transcription of the marORAB operon is inducible two to three fold by tetracycline, chloramphenicol, salicylate, and other structurally unrelated compounds (Cohen et al., supra). Activated cells become resistant not only to multiple unrelated antibiotics but also to oxidative stress agents and organic solvents (Cohen et al., supra; George, et al. (1983) J. Bacteriol. 155:531-540; George et al. (1996) FEMS Micro. Let. 139:1-10).
In the presence of selective agents (e.g., tetracycline, chloramphenicol, nalidixic acid, rifampin, penicillin, and cephalosporin) Mar mutants arise spontaneously at a frequency of 10xe2x88x927 (George, et al., J. Bacteriol. supra). Such mutants have been reported to favor the accumulation of secondary mutations leading to the expression of higher levels of resistance to novel or improved antimicrobial agents. For example, Mar mutants have recently been found among fluoroquinolone-resistant clinical isolates of E. coli (Maneewannakul et al.(996) Antimicrob. Agents Chemo. 38:542-546).
Characterization of several Mar mutant resistant strains has revealed constitutive transcription of mRNA from the marORAB operon as a result of various mutations within that operon (Cohen et al., supra). Consistent with these findings, the disruption of the mar locus has correlated with the complete loss of resistance. The resistance phenotype has been completely reversed to susceptibility by insertion of Tn5, a transposon element, into the marA gene of the E. coli chromosome. (George et al. (1983) J. Bacteriol. 155:541-548).
A promising new approach to antimicrobial therapy lies in the use of short synthetic strands of nucleic acids, called antisense oligonucleotides, to control gene expression. Inhibition of gene expression by antisense oligonucleotides relies at least in part, on the ability of the oligonucleotide to bind a complementary messenger RNA sequence, thereby preventing its translation (see generally, Agrawal (1992) Trends in Biotech. 10:152; Wagner et al.(1994) Nature 372:333-335; and Stein et al. (1993) Science 261:1004-1012). Synthetic oligonucleotides administered exogenously compose an alternate class of therapeutic agents and have been used successfully in both prokaryotic and eucaryotic systems.
Antisense oligonucleotides have been developed as antiparasitic agents, although none have been demonstrated to reverse the drug resistant phenotype of a drug resistant parasite strain. PCT publication No. WO 93/13740 discloses the use of antisense oligonucleotides directed to nucleic acids encoding the dihydrofolate reductase-thymidilate synthase gene of P. falciparum to inhibit propagation of drug-resistant malarial parasites. Rapaport et al. (Proc. Natl. Acad Sci. (USA) (1992) 89:8577-8580) teaches inhibition of the growth of chloroquine-resistant and chloroquine-sensitive P. falciparum in vitro using oligonucleotides directed to the dihydrofolate reductase-thymidylate synthase gene. PCT publication No. WO 94/12643 discloses antisense oligonucleotides directed to nucleic acids encoding a carbamoyl phosphate synthetase of P. falciparum. Tao et al. (Antisense Res. Dev. (1995) 5:123-129) teaches inhibition of propagation of a schistosome parasite by antisense oligonucleotides. Early experiments by Jayaraman showed that an antisense oligonucleotide complementary to the Shine-Delagarno ribosomal docking sequence of E. coli 16S rRNA, inhibited translation of bacterial mRNA in cell-free extracts derived from E. coli (Jayaraman (1996) Proc. Natl. Acad. Sci (USA) 93:709-713). Furthermore, experiments were conducted by Gasparro et al. using a photoactivatable antisense DNA construct to suppress ampicillin resistance in E. coli (Gasparro et al. Antisense Res. Dev. (1991) 1:117-140). More recently, experiments utilizing antisense oligodeoxyribonucleotide phosphorothioates have shown the successful inhibition of the growth of a wild-type and drug resistant strain of Mycobacterium smegmatis (Rapaport et al. (1996) Proc. Natl. Acad. Sci. (USA) 93:709-713).
Bacteria have been known to mutate extensively, resulting in a large number of strains which have become resistant to most drugs presently available. In addition, new resistant bacterial strains are likely to develop as the time progresses. Thus, there is a continued need for development of additional therapeutic agents and effective methods to treat these bacterial infections. Inactivation or suppression of the multiple antibiotic resistance operon would ideally make some prokaryotes more susceptible to a larger number of antimicrobial agents and environmental stresses, thus providing novel means to counter increased bacterial resistance.
The invention disclosed herein satisfies this need. The present inventors have discovered that antisense oligonucleotides that are complementary to sequences found in the marORAB operon can inhibit or suppress the expression of one or more genes within that operon, thereby making bacteria more susceptible to a larger number of antimicrobial agents and environmental stresses.
This discovery has been exploited to develop the present invention which includes antisense oligonucleotides directed to mRNA derived from the marORAB operon sequences which specifically inhibit or suppress the expression of one or more genes within that operon, and which are therefore useful both as therapeutic agents and as tools to elucidate the role and biological significance of the marORAB operon sequences.
In one aspect, the invention provides a synthetic oligonucleotide complementary to a transcript of the marORAB operon which inhibits the expression of a gene in the operon.
As used herein, the term xe2x80x9coperonxe2x80x9d is a unit of bacterial gene expression and regulation. Typically an operon includes nucleic acid and control elements in the nucleic acid which may be recognized by regulators of gene products. In the case of marORAB operon, the nucleic acid includes a regulatory region, designated marO, containing a promoter and an AUG start codon for rightward transcription of the loci designated marR, marA and marB.
For purposes of the invention, the term xe2x80x9ctranscriptxe2x80x9d is used to refer to ribonucleic acid transcribed from DNA, some of which is capable of serving as a substrate for the translation of one or more peptide products.
As used herein, the term xe2x80x9clocusxe2x80x9d refers to a position on a chromosome at which nucleic acid encoding a particular gene or genes reside. The nucleic acid comprises a start codon and at least one codon encoding an amino acid residue. Typically, a locus is transcribed to produce at least one mRNA transcript which in turn may be translated into a peptide.
As used herein, the term xe2x80x9csynthetic oligonucleotidexe2x80x9d refers to chemically synthesized polymers of nucleotides covalently attached by internucleotide linkages. The term xe2x80x9cinternucleotide linkagexe2x80x9d is used to refer to the covalent bonding between nucleotides which are thus attached via at least one 5xe2x80x2 to 3xe2x80x2 internucleotide linkage.
In some embodiments of the invention the oligonucleotide contains at least one internucleotide linkage selected from the group consisting of alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. In some other embodiments, the oligonucleotide contains, in addition to an internucleotide linkage selected from the linkages recited, at least one phosphothioate internucleotide linkage.
In some embodiments, the oligonucleotides contain at least one deoxyribonucleotide, at least a ribonucleotide, or both deoxyribonucleotide(s) and ribonucleotide(s).
In some embodiments, the synthetic oligonucleotide is from about 15 to about 50 nucleotides in length. In preferred embodiments, these oligonucleotides contain from about 15 to about 21 nucleotides.
In some particular embodiments of the invention, marORAB oligonucleotides have a nucleotide sequence selected from the group consisting of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5, and NO:6.
Another aspect of the invention provides a method of inhibiting the expression of the marORAB operon comprising the step of contacting a transcript of marORAB operon with a synthetic oligonucleotide complementary to the transcript. In some embodiments of the invention, the oligonucleotide is complementary to a locus selected from the group consisting of marO, marO/R, marR, marR/A, and marA. In yet other embodiments of the invention, the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5, and NO:6.
An additional aspect of the invention provides a method of reducing bacterial resistance to an antibiotic by exposing a resistant bacterium to synthetic oligonucleotide complementary to a transcript of the marORAB operon. The bacterium further may be treated with an antibiotic.
The term xe2x80x9cantibioticxe2x80x9d is art recognized and includes a composition which decreases the viability or which inhibits the growth or reproduction of microorganisms. As used in this disclosure, an antibiotic is further intended to include an antimicrobial or bactericidal agent.
In an additional embodiment of the invention the oligonucleotide is complementary to a transcript of a locus selected from the group consisting of marO, marO/R, marR, marR/A, and marA. In yet another embodiment of the invention, the oligonucleotide comprises a nucleotide sequence selected from the group cc ns sting of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5, and NO:6.
In an additional embodiment, bacteria are contacted with at least two synthetic oligonucleotides selected from the group consisting of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5, and NO:6.
Another aspect of the invention pertains to a method of treating a bacterial infection in a subject by administering to the subject a therapeutic amount of a synthetic oligonucleotide complementary to a transcript of the marORAB operon which is effective in reducing bacterial resistance to antibacterial agent.
In one embodiment of the invention, the oligonucleotide is complementary to a transcript of a gene or locus selected from the group consisting of marO, marO/R, marR, marR/A, and marA. In an additional embodiment of the invention the oligonucleotide includes a nucleotide sequence selected from the group consisting of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5, and NO:6.
An additional aspect of the invention is a pharmaceutically acceptable composition comprising a synthetic oligonucleotide complementary to a transcript of marORAB operon nucleic acid which inhibits the expression of one or more genes in the operon, and a physiologically acceptable carrier.
As used herein, the term xe2x80x9cpharmaceutically acceptablexe2x80x9d means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The term xe2x80x9cphysiologically acceptablexe2x80x9d refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
In one embodiment of the invention, the oligonucleotide is complementary to a locus selected from the group consisting of marO, marO/R, marR, marR/A, and marA.
In yet another embodiment, the oligonucleotide consists essentially of SEQ ID NO:1, NO:2, NO:3, NO:4, NO:5, and NO:6.